This proposal is directed at a better understanding of the mechanisms by which hyperglycemia affects angiogenesis in diabetes-associated vascular disease. Cardiovascular disease constitutes the major cause of mortality and morbidity in diabetes patients. Clinical and experimental studies revealed that hyperglycemia is the primary factor in the pathogenesis of diabetic vascular complications. However, the molecular mechanisms responsible for these effects are not fully understood. Accumulating evidence indicates that increased glucose flux through the hexosamine biosynthesis pathway (HBP) participates in mediating the effects of hyperglycemia. It has been proposed that the effects of the HBP are mediated by the O-linked N-acetylglucosamine (O-GlcNAc) modification on protein serine and threonine residues. Like phosphorylation, this O-GlcNAc modification is dynamic and inducible, and has been demonstrated to regulate target protein function and affect transcription, translation, and signaling. Because of the central role of angiogenesis in diabetic vascular complications, we propose to focus on the effects of protein O-GlcNAc modification induced by excess HBP flux on this process. Our preliminary data shown in this application suggest that protein O-GlcNAc modification impairs angiogenesis in cultured endothelial cells. Our current hypothesis is that in the presence of chronic hyperglycemia, activation of the HBP inhibits the new blood vessel formation by O-GlcNAc modification of key angiogenic signaling proteins in endothelial cells. Our Specific Aims are: 1) Investigate the impact of O-GlcNAc on angiogenesis in endothelial cells. We will examine the effects of O-GlcNAc by in vitro and in vivo angiogenesis assays, and further assess the effect of hyperglycemia on angiogenesis in diabetic mice. 2) Identify the mechanisms of angiogenesis regulation by O-GlcNAc. Gene transcription profile and proteomic analysis will be performed to identify mediators for angiogenesis regulation by O-GlcNAc. 3) Determine if upregulation of HBP in the intact animal affects angiogenesis. Atransgenic animal model, wherein higher HBP is induced specifically in endothelial cells, will be generated and a series of angiogenesis assays will be performed to examine the phenotypes. Thus, these approaches will elucidate the molecular mechanism of how hyperglycemia contributes to the development of vascular disease in diabetes and may provide new therapeutic strategies for treatment of diabetic vascular complications. [unreadable] [unreadable] [unreadable]